多铜氧化酶／中介体催化氧还原反应动力学分析

Kinetic Analysis of Oxygen Reduction Reaction Catalyzed by Multi-copper Oxidase in Presence of Electron Relay

采用分光光度法、Clark型氧电极以及循环伏安法结合旋转圆盘电极技术,分别测定了游离多铜氧化酶在扩散型电子中介体存在时,催化氧还原循环中每一个组成步骤的速率并进行了比较,试图确定这个催化反应的决速步骤。实验结果表明,漆酶分子内部的电子迁移速率(103/s)最高,酶催化氧气化学还原的速率次之(91/s),酶催化中介体氧化产物在电极上电化学还原的速率再次之(0.19/s或7.8×10-3cm/s),底物O2气以及氧化态/还原态电子中介体2,2'-连氮-双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)的传质系数分别为1.7×10-3、4.4×10-4和6.3×10-4cm/s,相应地酶催化中介体氧化的化学反应速率为0.047/s,酶催化中介体氧化的化学反应步骤以及中介体的传质步骤是影响催化反应速率的关键。在此基础上,通过系统改变体系中酶的种类、活力以及浓度、中介体种类及浓度、溶液温度及pH值等参数,研究了酶电催化氧还原活力与这些参数之间的依赖关系,进一步确证了前述的结论。

Rates of each procedure composed of catalytic oxygen reduction reaction cycle with free multicopper oxidase as electro-catalyst in the presence of diffusional electron relay were determined by the spectroscopy, Clark-type oxygen electrode and cyclic voltammetry combined with rotating disc electrode technique, respectively. These rates were then compared to verify the rate-determining step in the catalytic reaction. Results from experiments indicate that the fastest step is the internal electron transfer in enzyme molecule（103/s） followed by the enzymatic catalysis of oxygen transformation into water（91/s）. The third procedure as for the rate was attributed to the electrochemical reduction reaction of the oxidation product of enzymatic catalytic reaction for electron mediator （0. 19/s or 7. 8 × 10 ^-3 cm/s ） at the surface of base electrode. Mass transfer coefficients of the substrate, oxygen molecule, and oxidized/reduced electron mediator, 2,2＇-azino-bis-（3-ethylbenzthiazoline-6-sulfonic acid） diammonium（ ABTS）, were evaluated to be 1.7 × 10^-3 cm/s, 4. 4 × 10^-4 cm/s and 6. 3 × 10^-4 cm/s, respectively. Accordingly, chemical reaction rate in the enzyme catalytic electron mediator oxidation was determined to be 0. 047/s. So it is apparent that the key procedure of the whole enzymatic catalysis cycle should be ascribed to the enzyme catalytic oxidation of electron mediator and the mass transfer of electron relay. enzyme, specific activity of enzyme, enzyme concentration, and pH value of electrolyte, etc. ） on the enzyme investigated according to systematic modulation of conclusions from the kinetic analysis. The influence of parameters （including sorts of types of mediators and their contents, temperature electro-catalyzed oxygen reduction performance was those parameters. The results confirmed the above.